Methods for analysis of viral capsid protein composition

ABSTRACT

Methods of determining the stoichiometry of a viral capsid and/or determining the heterogeneity of protein components in a viral capsid are disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 USC § 119(e) of U.S.Provisional Application No. 62/750,583, filed Oct. 25, 2018, which isincorporated herein by reference in its entirety for all purposes.

FIELD OF THE INVENTION

The present invention pertains to methods for determining theheterogeneity of a viral particle, such as an adeno-associated virus(AAV) particle using hydrophilic interaction liquid chromatography(HILIC) and mass spectrometry determination.

BACKGROUND

Gene therapy has emerged as an alternative treatment for geneticdiseases. Gene therapy involves the transfer of some genetic material(DNA, RNA or oligonucleotides) into target cells. In practice, the geneof interest (also called a transgene) must be delivered to the cell by avector, which carries a molecule of DNA or RNA. It is based on thetransfer of functional genes to replace or supplement defective genes.The transgene can be delivered into the cell by the vector. The methodof delivery differs depending on the type of treatment and organ/tissueto be targeted.

Viral particles have emerged as vectors for gene therapy and thetreatment of disease. Viral vectors, such as those based on the genomeof adeno-associated virus (AAV), offer exciting platforms for genedelivery. Currently, 12 human serotypes of AAV (AAV1-12) have beendescribed, many of which have distinct cell and tissue tropism,potentially creating the option to generate a variety of differentvector classes from this viral genus.

However, one of the problems facing the adoption of viral vectors ingene therapy is the characterization of viral particle homogeneity.While classic techniques such as electron microscopy and Southern Blotscan characterize viral particle heterogeneity, such as AAV heterogeneityand aggregation, they do not provide sufficient resolution forquantifying homogeneity when it comes to producing clinical-grade viralvector preparations. Complete characterization of the constituent viralcapsid proteins, such as the capsid proteins of AAV vectors, includingtheir sequences and post-translational modifications (PTMs), is highlyrecommended to ensure product quality and consistency. Thus, methods areneeded to determine the homogeneity of viral particles.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides a method of determiningthe stoichiometry of protein components of a viral capsid of a viralparticle, in which the method comprises: (a) subjecting a sample ofviral particles to hydrophilic interaction liquid chromatography (HILIC)to separate the protein components of the viral capsid of the viralparticles; (b) determining the masses of protein components of the viralcapsid to identify the protein components separated by HILIC; and (c)determining the relative abundance of the protein components of theviral capsid from the HILIC separation, thereby determining thestoichiometry of protein components of a viral capsid of a viralparticle.

In another aspect, the present invention provides a method ofdetermining the heterogeneity of proteins in a capsid of a viralparticle, in which the method comprises: (a) subjecting the viralparticle to HILIC to separate protein components of the viral particlecapsid; (b) determining the masses of protein components of the proteincapsid; and (c) comparing the determined masses of the proteincomponents of the viral particle capsid with theoretical masses, whereina deviation of one or more of the masses of protein components of theviral particle capsid from the theoretical masses is indicative of thecapsid heterogeneity.

In some embodiments, the viral particle comprises an adeno-associatedvirus (AAV) particle.

In some embodiments, the protein components of the viral capsid compriseVP1, VP2 and VP3 of the AAV particle.

In some embodiments, the heterogeneity comprises one or more of mixedserotypes, variant capsids, capsid amino acid substitutions, truncatedcapsids, or modified capsids.

In some embodiments, the AAV particle comprises an AAV1 capsid, an AAV2capsid, an AAV3 capsid, an AAV4 capsid, an AAV5 capsid, an AAV6 capsid,an AAV7 capsid, an AAV8 capsid, an AAVrh8 capsid, an AAV9 capsid, anAAV10 capsid, an AAV11 capsid, an AAV 12 capsid, or a variant thereof.

In some embodiments, the masses of VP1, VP2, and VP3 are compared totheoretical masses of one or more of an AAV1 capsid, an AAV2 capsid, anAAV3 capsid, an AAV4 capsid, an AAV5 capsid, an AAV6 capsid, an AAV7capsid, an AAV8 capsid, AAVrh8 capsid, an AAV9 capsid, an AAV10 capsid,an AAV11 capsid, an AAV 12 capsid, or a variant thereof.

In some embodiments, the AAV particle comprises an AAV1 invertedterminal repeat sequence (ITR), an AAV2 ITR, an AAV3 ITR, an AAV4 ITR,an AAV5 ITR, an AAV6 ITR, an AAV7 ITR, an AAV8 ITR, an AAVrh8 ITR, anAAV9 ITR, an AAV 10 ITR, an AAVrh10 ITR, an AAV11 ITR, or an AAV 12 ITR.

In some embodiments, the AAV particle has a capsid serotype selected fortransduction of cells of a subject's liver.

In some embodiments, the AAV particle is a recombinant AAV (rAAV)particle.

In some embodiments, the AAV particle comprises an AAV vector encoding aheterologous transgene.

In some embodiments, the AAV particle has a capsid serotype AAV7, AAV8,or AAV9.

In some embodiments, the AAV particle has a capsid serotype AAV9.

In some embodiments, the AAV particle has a capsid serotype AAV9 and isa viral vector encoding Lysosomal Alpha Glucosidase (GAA) linked to ananti-CD63 antibody.

In some embodiments, the viral particle comprises a viral vectorencoding a heterologous transgene.

In some embodiments, the viral particle belongs to a viral familyselected from the group consisting of Adenoviridae, Parvoviridae,Retroviridae, Baculoviridae, and Herpesviridae.

In some embodiments, the viral particle belongs to a viral genusselected from the group consisting of Atadenovirus, Aviadenovirus,Ichtadenovirus, Mastadenovirus, Siadenovirus, Ambidensovirus,Brevidensovirus, Hepandensovirus, Iteradensovirus, Penstyldensovirus,Amdoparvovirus, Aveparvovirus, Bocaparvovirus, Copiparvovirus,Dependoparvovirus, Erythroparvovirus, Protoparvovirus, Tetraparvovirus,Alpharetrovirus, Betaretrovirus, Deltaretrovirus, Epsilonretrovirus,Gammaretrovirus, Lentivirus, Spumavirus, Alphabaculovirus,Betabaculovirus, Deltabaculovirus, Gammabaculovirus, Iltovirus,Mardivirus, Simplexvirus, Varicellovirus, Cytomegalovirus,Muromegalovirus, Proboscivirus, Roseolovirus, Lymphocryptovirus,Macavirus, Percavirus, and Rhadinovirus.

In some embodiments, the Retroviridae is Moloney murine sarcoma virus(MoMSV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumorvirus (MuMTV), gibbon ape leukemia virus (GaLV), feline leukemia virus(FLV), spumavirus, Friend virus, Murine Stem Cell Virus (MSCV) RousSarcoma Virus (RSV), human T cell leukemia viruses, HumanImmunodeficiency Virus (HIV), feline immunodeficiency virus (FIV),equine immunodeficiency virus (EIV), visna-maedi virus; caprinearthritis-encephalitis virus; equine infectious anemia virus; felineimmunodeficiency virus (FIV); bovine immune deficiency virus (BIV); orsimian immunodeficiency virus (SIV).

In some embodiments, the HILIC uses a mobile phase A comprisingtrifluoroacetic acid in water.

In some embodiments, the mobile phase A comprises about 0.1%trifluoroacetic acid.

In some embodiments, the chromatography comprises a mobile phase Bcomprising trifluoroacetic acid in acetonitrile.

In some embodiments, the mobile phase B comprises about 0.1%trifluoroacetic acid.

In some embodiments, the proportion of mobile phase A in thechromatography increases over time.

In some embodiments, the mobile phase A increases from about 15% toabout 100%, over about 45 minutes.

DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic model of a possible treatment regimen for Pompedisease that includes the use of a Adeno-Associated Virus (AAV) as aVector for Gene Therapy.

FIG. 2A is a model of an AAV viral capsid.

FIG. 2B is schematic representation of the viral capsid proteins from anAAV serotype and their approximate masses.

FIG. 3A is UV trace from a reverse phase TIC separation of AAV capsidproteins from an AAV particle showing poor resolution of the individualproteins.

FIG. 3B is a comparison of several prior art separation methods showingboth poor resolution and/or poor quantification.

FIG. 4 is UV trace from a hydrophilic interaction liquid chromatography(HILIC) separation of AAV capsid proteins from an AAV particle showinghigh resolution of the individual proteins and the determination of therelative abundance of the individual proteins.

FIGS. 5A-5D are mass spectra of AAV capsid proteins from an AAVparticle.

FIG. 6 shows two traces of HILIC-UV analysis of AAV9 viral particles andtheir stoichiometry determination.

DETAILED DESCRIPTION OF THE INVENTION

Before the present invention is described, it is to be understood thatthis invention is not limited to particular methods and experimentalconditions described, as such methods and conditions may vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting, since the scope of the present invention will be limitedonly by the appended claims. Any embodiments or features of embodimentscan be combined with one another, and such combinations are expresslyencompassed within the scope of the present invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. As used herein, the term“about,” when used in reference to a particular recited numerical value,means that the value may vary from the recited value by no more than 1%.For example, as used herein, the expression “about 100” includes 99 and101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.)

Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of the presentinvention, the preferred methods and materials are now described. Allpatents, applications and non-patent publications mentioned in thisspecification are incorporated herein by reference in their entireties.

Abbreviations Used Herein

MS/MS: Tandem Mass Spectrometry

MS: Mass Spectrometry

ITRs: Inverted Terminal Repeat Sequences

rAAV vector: Recombinant AAV Vector

HILIC: Hydrophilic Interaction Liquid Chromatography

GAA: Lysosomal Alpha Glucosidase

mAb: Monoclonal Antibody

IgG: Immunoglobulin G

LC: Light Chain

HC: Heavy Chain

AAV: Adeno-Associated Virus

PTMs: Post-translational Modifications

ERT: enzyme replacement therapy

Definitions

“Adeno-associated virus” or “AAV”: AAV is a non-pathogenic parvovirus,with single-stranded DNA, a genome of approximately 4.7 kb, notenveloped and has icosahedric conformation. AAV was first discovered in1965 as a contaminant of adenovirus preparations. AAV belongs to theDependovirus genus and Parvoviridae family, requiring helper functionsfrom either herpes virus or adenovirus for replication. In the absenceof helper virus, AAV can set up latency by integrating into humanchromosome 19 at the 19q13.4 location. The AAV genome consists of twoopen reading frames (ORF), one for each of two AAV genes, Rep and Cap.The AAV DNA ends have a 145-bp inverted terminal repeat (ITR), and the125 terminal bases are palindromic, leading to a characteristic T-shapedhairpin structure.

The Rep gene is transcribed from promoters p5 and p19 into four Repproteins (Rep78, Rep68, Rep52, and Rep40), which have important roles inthe life cycle of the virus. Proteins Rep78 and Rep68 are encoded by themRNA transcribed from promoter p5. These proteins are essential forviral DNA replication, transcription and control of site-specificintegration. The two smaller proteins Rep52 and Rep40 are generated bythe mRNA transcribed from promoter p19. These proteins are involved inthe formation of a single-stranded viral genome for packaging and viralintegration. The Cap gene encodes three viral capsid proteins: VP1 (735amino acids, ˜90 kDa), VP2 (598 amino acids, ˜72 kDa) and VP3 (533 aminoacids, ˜60 kDa), which form the viral capsid of 60 subunits, at theratio of 1:1:10 (see FIGS. 2A and 2B). The three capsid proteins aretranslated from the mRNA transcribed from the promoter p40.

The term “antibody”, as used herein, is intended to refer toimmunoglobulin molecules comprised of four polypeptide chains, two heavy(H) chains and two light (L) chains inter-connected by disulfide bonds(i.e., “full antibody molecules”), as well as multimers thereof (e.g.IgM) or antigen-binding fragments thereof. Each heavy chain is comprisedof a heavy chain variable region (“HCVR” or “V_(H)”) and a heavy chainconstant region (comprised of domains C_(H)1, C_(H)2 and C_(H)3). Invarious embodiments, the heavy chain may be an IgG isotype. In somecases, the heavy chain is selected from IgG1, IgG2, IgG3 or IgG4. Insome embodiments, the heavy chain is of isotype IgG1 or IgG4, optionallyincluding a chimeric hinge region of isotype IgG1/IgG2 or IgG4/IgG2.Each light chain is comprised of a light chain variable region (“LCVR or“V_(L)”) and a light chain constant region (C_(L)). The V_(H) and V_(L)regions can be further subdivided into regions of hypervariability,termed complementarity determining regions (CDR), interspersed withregions that are more conserved, termed framework regions (FR). EachV_(H) and V_(L) is composed of three CDRs and four FRs, arranged fromamino-terminus to carboxy-terminus in the following order: FR1, CDR1,FR2, CDR2, FR3, CDR3, FR4. The term “antibody” includes reference toboth glycosylated and non-glycosylated immunoglobulins of any isotype orsubclass. The term “antibody” includes antibody molecules prepared,expressed, created or isolated by recombinant means, such as antibodiesisolated from a host cell transfected to express the antibody. For areview on antibody structure, see Lefranc et al., IMGT unique numberingfor immunoglobulin and T cell receptor variable domains and Igsuperfamily V-like domains, 27(1) Dev. Comp. Immunol. 55-77 (2003); andM. Potter, Structural correlates of immunoglobulin diversity, 2(1) Surv.Immunol. Res. 27-42 (1983).

The term antibody also encompasses a “bispecific antibody”, whichincludes a heterotetrameric immunoglobulin that can bind to more thanone different epitope. One half of the bispecific antibody, whichincludes a single heavy chain and a single light chain and six CDRs,binds to one antigen or epitope, and the other half of the antibodybinds to a different antigen or epitope. In some cases, the bispecificantibody can bind the same antigen, but at different epitopes ornon-overlapping epitopes. In some cases, both halves of the bispecificantibody have identical light chains while retaining dual specificity.Bispecific antibodies are described generally in U.S. Patent App. Pub.No. 2010/0331527(Dec. 30, 2010).

The terms “antigen-binding portion” and “antigen-binding fragment” of anantibody (or “antibody fragment”), refers to one or more fragments of anantibody that retain the ability to specifically bind to an antigen.Examples of binding fragments encompassed within the term“antigen-binding portion” of an antibody include (i) a Fab fragment, amonovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) aF(ab′)2 fragment, a bivalent fragment comprising two Fab fragmentslinked by a disulfide bridge at the hinge region; (iii) a Fd fragmentconsisting of the VH and CH1 domains; (iv) a Fv fragment consisting ofthe VL and VH domains of a single arm of an antibody, (v) a dAb fragment(Ward et al. (1989) Nature 241:544-546), which consists of a VH domain,(vi) an isolated CDR, and (vii) an scFv, which consists of the twodomains of the Fv fragment, VL and VH, joined by a synthetic linker toform a single protein chain in which the VL and VH regions pair to formmonovalent molecules. Other forms of single chain antibodies, such asdiabodies are also encompassed under the term “antibody” (see e.g.,Holliger et at. (1993) 90 PNAS U.S.A. 6444-6448; and Poljak et at.(1994) 2 Structure 1121-1123).

Moreover, antibodies and antigen-binding fragments thereof can beobtained using standard recombinant DNA techniques commonly known in theart (see Sambrook et al., 1989).

The term “human antibody”, is intended to include antibodies havingvariable and constant regions derived from human germ lineimmunoglobulin sequences. The human mAbs of the invention may includeamino acid residues not encoded by human germline immunoglobulinsequences (e.g., mutations introduced by random or site-specificmutagenesis in vitro or by somatic mutation in vivo), for example in theCDRs and in particular CDR3. However, the term “human antibody”, as usedherein, is not intended to include mAbs in which CDR sequences derivedfrom the germline of another mammalian species (e.g., mouse), have beengrafted onto human FR sequences. The term includes antibodiesrecombinantly produced in a non-human mammal, or in cells of a non-humanmammal. The term is not intended to include antibodies isolated from orgenerated in a human subject.

The term “corresponding” is a relative term indicating similarity inposition, purpose or structure. A mass spectral signal due to aparticular peptide or protein is also referred to as a signalcorresponding to the peptide or protein. In certain embodiments, aparticular peptide sequence or set of amino acids, such as a protein,can be assigned to a corresponding peptide mass.

The term “isolated,” as used herein, refers to a biological component(such as a nucleic acid, peptide, protein, lipid, viral particle ormetabolite) that has been substantially separated, produced apart from,or purified away from other biological components in the cell of theorganism in which the component naturally occurs or is transgenicallyexpressed.

“Mass spectrometry” is a method wherein, a sample is analyzed bygenerating gas phase ions from the sample, which are then separatedaccording to their mass-to-charge ratio (m/z) and detected. Methods ofgenerating gas phase ions from a sample include electrospray ionization(ESI), matrix-assisted laser desorption-ionization (MALDI),surface-enhanced laser desorption-ionization (SELDI), chemicalionization, and electron-impact ionization (EI). Separation of ionsaccording to their m/z ratio can be accomplished with any type of massanalyzer, including quadrupole mass analyzers (Q), time-of-flight (TOF)mass analyzers, magnetic sector mass analyzers, 3D and linear ion traps(IT), orbitrap mass analyzer, Fourier-transform ion cyclotron resonance(FT-ICR) analyzers, and combinations thereof (for example, aquadrupole-time-of-flight analyzer, or Q-TOF analyzer). Prior toseparation, the sample may be subjected to one or more dimensions ofchromatographic separation, for example HILIC.

Tandem mass spectrometry or MS/MS is a technique to break down selectedions (precursor ions) into fragments (product ions). The fragments thenreveal aspects of the chemical structure of the precursor ion. In tandemmass spectrometry, once samples are ionized (for example by ESI, MALDI,EI, etc.) to generate a mixture of ions, precursor ions, for examplepeptides from a digest, of a specific mass-to-charge ratio (m/z) areselected (MS1) and then fragmented (MS2) to generate a product ions fordetection. Typical Tandem MS instruments include QqQ, QTOF, and hybridion trap/FTMS, etc. One example of an application of tandem massspectrometry is protein identification. The first mass analyzer isolatesions of a particular m/z value that represent a single species ofpeptide among many introduced into and then emerging from the ionsource. Those ions are then accelerated into a collision cell containingan inert gas such as argon to induce ion fragmentation. This process isdesignated collisionally induced dissociation (CID) or collisionallyactivated dissociation (CAD). The m/z values of fragment ions are thenmeasured in a 2^(nd) mass analyzer to obtain amino acid sequenceinformation. Tandem mass spectrometry can be used to identify thesequence of a peptide and hence full or partial length proteinsaccording to the methods disclosed herein. Precursor ions can beactivated (with increased internal energy) in many different ways.Fragmentation patterns depend on how energy is transferred to theprecursor ion, the amount of energy transferred, and how the transferredenergy is internally distributed. Collision-induced dissociation andinfrared multiphoton dissociation are “slow-heating” techniques thatincrease the Boltzmann temperature of the ion and thus preferentiallycleave the weakest bonds.

The terms “peptide,” “protein” and “polypeptide” refer, interchangeably,to a polymer of amino acids and/or amino acid analogs that are joined bypeptide bonds or peptide bond mimetics. The twenty naturally-occurringamino acids and their single-letter and three-letter designations are asfollows: Alanine A Ala; Cysteine C Cys; Aspartic Acid D Asp; Glutamicacid E Glu; Phenylalanine F Phe; Glycine G Gly; Histidine H His;Isoleucine I He; Lysine K Lys; Leucine L Leu; Methionine M Met;Asparagine N Asn; Proline P Pro; Glutamine Q Gln; Arginine R Arg; SerineS Ser; Threonine T Thr; Valine V Val; Tryptophan w Trp; and Tyrosine YTyr.

References to a mass of an amino acid means the monoisotopic mass oraverage mass of an amino acid at a given isotopic abundance, such as anatural abundance. In some examples, the mass of an amino acid can beskewed, for example, by labeling an amino acid with an isotope. Somedegree of variability around the average mass of an amino acid isexpected for individual single amino acids based on the exact isotopiccomposition of the amino acid. The masses, including monoisotopic andaverage masses for amino acids are easily obtainable by one of ordinaryskill the art.

Similarly, references to a mass of a peptide or protein means themonoisotopic mass or average mass of a peptide or protein at a givenisotopic abundance, such as a natural abundance. In some examples, themass of a peptide can be skewed, for example, by labeling one or moreamino acids in the peptide or protein with an isotope. Some degree ofvariability around the average mass of a peptide is expected forindividual single peptides based on the exact isotopic composition ofthe peptide. The mass of a particular peptide can be determined by oneof ordinary skill the art.

A “vector,” as used herein, refers to a recombinant plasmid or virusthat comprises a nucleic acid to be delivered into a host cell, eitherin vitro or in vivo.

The term “polynucleotide” or “nucleic acid” as used herein refers to apolymeric form of nucleotides of any length, either ribonucleotides ordeoxyribonucleotides. Thus, this term includes, but is not limited to,single-, double- or multi-stranded DNA or RNA, genomic DNA, cDNA,DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases, orother natural, chemically or biochemically modified, non-natural, orderivatized nucleotide bases. The backbone of the nucleic acid cancomprise sugars and phosphate groups (as may typically be found in RNAor DNA), or modified or substituted sugar or phosphate groups.

Alternatively, the backbone of the nucleic acid can comprise a polymerof synthetic subunits such as phosphoramidates and thus can be anoligodeoxynucleoside phosphoramidate (P-NH2) or a mixedphosphoramidate-phosphodiester oligomer. In addition, a double-strandednucleic acid can be obtained from the single stranded polynucleotideproduct of chemical synthesis either by synthesizing the complementarystrand and annealing the strands under appropriate conditions, or bysynthesizing the complementary strand de novo using a DNA polymerasewith an appropriate primer.

A “recombinant viral vector” refers to a recombinant polynucleotidevector including one or more heterologous sequences (i.e., nucleic acidsequence not of viral origin).

A “recombinant AAV vector (rAAV vector)” refers to a polynucleotidevector including one or more heterologous sequences (i.e., nucleic acidsequence not of AAV origin) that may be flanked by at least one, e.g.,two, AAV inverted terminal repeat sequences (ITRs). Such rAAV vectorscan be replicated and packaged into infectious viral particles whenpresent in a host cell that has been infected with a suitable helpervirus (or that is expressing suitable helper functions) and that isexpressing AAV rep and cap gene products (i.e. AAV Rep and Capproteins).

A “viral particle” refers to a viral particle composed of at least oneviral capsid protein and an encapsulated viral genome.

“Heterologous” means derived from a genotypically distinct entity fromthat of the rest of the entity to which it is compared or into which itis introduced or incorporated. For example, a nucleic acid introduced bygenetic engineering techniques into a different cell type is aheterologous nucleic acid (and, when expressed, can encode aheterologous polypeptide). Similarly, a cellular sequence (e.g., a geneor portion thereof) that is incorporated into a viral vector is aheterologous nucleotide sequence with respect to the vector.

An “inverted terminal repeat” or “ITR” sequence is relatively shortsequences found at the termini of viral genomes which are in oppositeorientation. An “AAV inverted terminal repeat (ITR)” sequence, is anapproximately 145-nucleotide sequence that is present at both termini ofa single-stranded AAV genome.

The term “hydrophilic interaction chromatography” or HILIC is intendedto include a process employing a hydrophilic stationary phase and ahydrophobic organic mobile phase in which hydrophilic compounds areretained longer than hydrophobic compounds. In certain embodiments, theprocess utilizes a water-miscible solvent mobile phase.

The term “sample,” as used herein, refers to a mixture of molecules thatcomprises at least a viral particle, such as an AAV particle, that issubjected to manipulation in accordance with the methods of theinvention, including, for example, separating, analyzing, extracting,concentrating, profiling and the like.

The term “chromatographic surface,” as used herein, includes a surfacewhich is exposed to a sample or analytes. A chromatographic surface canbe chemically modified, functionalized or activated or have amicrostructure, e.g. a pore. In certain embodiments, the chromatographicsurface can be hydrophobic, hydrophilic (polar) or ionic. In otherembodiments, the chromatographic surface is fully porous, superficiallyporous or non-porous.

The term “chromatographic core,” as used herein, includes achromatographic material, including but not limited to an organicmaterial such as silica, in the form of a particle, a monolith oranother suitable structure, which forms an internal portion of thematerials of the invention. In certain aspects, the surface of thechromatographic core represents the chromatographic surface, orrepresents a material encased by a chromatographic surface, as definedherein. The chromatographic surface material may be disposed on orbonded to or annealed to the chromatographic core in such a way that adiscrete or distinct transition is discernible or may be bound to thechromatographic core in such a way as to blend with the surface of thechromatographic core resulting in a gradation of materials and nodiscrete internal core surface. In certain aspects, the chromatographicsurface material may be the same or different from the material of thechromatographic core and may exhibit different physical orphysiochemical properties from the chromatographic core, including, butnot limited to, pore volume, surface area, average pore diameter, carboncontent or hydrolytic pH stability.

The term “hydrophilic,” as used herein, describes having an affinityfor, attracting, adsorbing or absorbing water.

The term “hydrophobic,” as used herein, describes lacking an affinityfor, repelling, or failing to adsorb or absorb water.

“Chromatography,” as used herein, refers to the process of separating amixture, for example a mixture containing viral capsid proteins. Itinvolves passing a mixture through a stationary phase, which separatesmolecules of interest from other molecules in the mixture and allows oneor more molecules of interest to be isolated. An example of a method ofchromatographic separation is hydrophilic interaction liquidchromatography (HILIC).

“Contacting,” as used herein, includes bringing together at least twosubstances in solution or solid phase, for example contacting astationary phase of a chromatography material with a sample, such as asample comprising viral particles.

General Description

Pompe disease is an autosomal recessive lysosomal storage disordercaused by mutations in the GAA gene encoding acid α-glucosidase (GAA)—alysosomal enzyme responsible for the hydrolysis of glycogen to glucose.Deficiency in GAA results in accumulation of glycogen in lysosomes andsubsequent cellular dysfunction in cardiac, skeletal, and smooth musclesas well as in the central nervous system. Pompe disease can presentearly in life as infantile onset Pompe disease (IOPD) or later inchildhood to adulthood as late onset Pompe disease (LOPD). Respiratoryfailure is a prominent cause of death in both types of Pompe disease.

Currently, the only Food and Drug Administration approved treatment forPompe disease is enzyme replacement therapy (ERT). However, systemicallyadministered GAA does not cross the blood-brain barrier and thereforecannot treat the CNS pathology and affected respiratory motor neurons.Furthermore, ERT only partially corrects skeletal muscle abnormalitiesas a result of low uptake into muscle fibers. Consequently, two-thirdsof IOPD patients eventually require ventilator support, and respiratoryinsufficiency persists among LOPD patients

Gene therapy using adeno-associated virus (AAV) vectors is ideal forPompe disease, since it is a monogenetic disorder. One of the strategiesthat is being studied is the combination of enzyme replacement withlinked antibodies. In one example, high expression of anti-CD63::GAAfrom the liver via gene therapy is being developed to overcome theimmune response to replacement enzyme seen in patients with noendogenous enzyme (see FIG. 1). As with all viral vector systems, it isimportant to insure that the therapeutic composition contains the rightamount of correctly formed viral particles. Thus, determiningstoichiometry and protein composition of viral particles is veryimportant

Aspects of this disclosure are directed to a method of determining thestoichiometry of protein components of a viral capsid of a viralparticle. In embodiments, the method includes subjection a sample ofviral particles to hydrophilic interaction liquid chromatography (HILIC)to separate the protein components of the viral capsid of the viralparticles, such as viral particles of interest where information aboutthe capsid is desired. In embodiments, an HILIC column is contacted withthe sample containing the viral particles. In certain embodiments themethod includes determining the masses of protein components of theviral capsid to identify the protein components separated by HILIC, forexample, using mass spectrometry techniques, such as those describedherein. In embodiments, the method includes calculating the relativeabundance of the protein components of the viral capsid from the HILICseparation to determine the stoichiometry of protein components of aviral capsid of a viral particle, for example using ultraviolet (UV)detection of the protein components of the viral capsid as they areeluted from the HILIC column. For example, the area of a UV peak can beused to determine the relative abundance of the capsid proteins and usedto calculate the stoichiometry of the capsid proteins in the vitalcapsid (see, FIG. 4). In another example, the peak height and/or peak UVintensity is used to determine relative abundance. In some embodiments,the retention time of the different proteins on the HILIC column isdetermined as a function of the mobile phase used and, in subsequentanalysis this retention time can be used to determine the proteins andrelative abundance of the proteins from the viral particle without theneed to determine the mass and/or identity of the proteins every time adetermination of stoichiometry is made, e.g. a standard value or valuescan be developed. Prior to this disclosure it was very difficult toresolve the different capsid proteins using conventional chromatographytechniques (see FIGS. 3A and 3B). Using the sample conditions discussedherein for HILIC, good separation was achieved for AAV viral particles.In addition, the use of the HILIC column removed any requirement for adenaturation step. In certain embodiments, the method is used todetermine the serotype of a viral particle. For example, the masses ofVP 1, VP2 and VP3 of each AAV serotype are unique and can be used toidentify or differentiate AAV capsid serotypes. In addition, theseparated capsid proteins can be subjected to downstream analysis, suchas a determination of protein sequence and post-translationalmodifications of the capsid proteins, for example with accurate massmeasurement at the intact protein level.

Aspects of this disclosure are directed a method of determining theheterogeneity of protein components in a capsid of a viral particle. Inembodiments, the method includes subjecting the viral particle to HILICto separate protein components of the viral particle capsid. Inembodiments, the method includes determining the masses of proteincomponents of the protein capsid. In some cases, the masses of theprotein components of the viral particle capsid are compared withtheoretical masses of the viral particle capsid. A deviation of one ormore of the masses of protein components of the viral particle capsidindicates that one or more proteins of the capsid are heterogeneous (seeFIG. 5A). Conversely, no deviation would indicate that the proteins ofthe capsid are homogeneous (see FIG. 5B-5D). In embodiments,heterogeneity is due to one or more of mixed serotypes, variant capsids,capsid amino acid substitutions, truncated capsids, or modified capsids.In some embodiments, the determination of the stoichiometry of proteincomponents of a viral capsid of a viral particle and the determinationof the heterogeneity of protein components in a capsid of a viralparticle are done on the same sample, for example is a single test.

In certain embodiments, the viral particle is an adeno-associated virus(AAV) particle and the methods disclosed can be used to determine thestoichiometry of protein components in a capsid of an AAV particleand/or heterogeneity of protein components in a capsid of a AAVparticle. In embodiments, the protein components of the protein capsidcomprise VP1, VP2 and VP3 of an AAV particle. In embodiments, the AAVparticle is a recombinant AAV (rAAV) particle. In embodiments, the AAVparticle includes an AAV vector encoding a heterologous transgene. Insome embodiments, a determined or calculated mass of the presentdisclosure (e.g., the determined or calculated mass of VP1, VP2 and/orVP3 of the AAV particle) may be compared with a reference, for example,a theoretical mass of a VP1 , VP2, and/or VP3 of one or more AAVserotypes (see, for example, FIGS. 2A and 2B). A reference may include atheoretical mass of a VP1, VP2, and/or VP3 of one or more of any of theAAV serotypes. For example, in some embodiments, the masses of VP1, VP2,and/or VP3 are compared to theoretical masses of one or more of an AAV1capsid, an AAV2 capsid, an AAV3 capsid, an AAV4 capsid, an AAV5 capsid,an AAV6 capsid, an AAV7 capsid, an AAV8 capsid, an AAVrh8 capsid, anAAV9 capsid, an AAV 10 capsid, an AAV 11 capsid, an AAV 12 capsid, or avariant thereof. In some embodiments, a determined or calculated mass(e.g., the determined or calculated mass of VP1, VP2 and/or VP3 of theAAV particle) may be compared with a theoretical mass of a VP1, VP2,and/or VP3 of the corresponding AAV serotype.

In some embodiments, the methods of the present disclosure may includedetermining the heterogeneity of the proteins of an AAV particle. Insome embodiments, a deviation of one or more of the masses of VP1, VP2and/or VP3 (e.g., from a reference mass, such as a theoretical,predicted, or expected mass) is indicative of the AAV capsid proteinheterogeneity. In some embodiments, heterogeneity may include one ormore of the following, without limitation: mixed serotypes, variantcapsids, capsid amino acid substitutions, truncated capsids, or modifiedcapsids.

In some embodiments, a method of determining the heterogeneity of an AAVparticle may include subjecting a denatured AAV particle to LC/MS (e.g.,as described herein), determining the masses of VP1, VP2 and VP3 of theAAV particle, and comparing these masses with theoretical masses of VP1,VP2 and VP3 of the AAV serotype; as well as subjecting fragments of VP1,VP2 and/or VP3 to LC/MS/MS, determining the masses of fragments of VP1,VP2 and VP3 of the AAV particle, and comparing these masses withtheoretical masses of VP1, VP2 and VP3 of the AAV serotype (a deviationof one or more of the masses of VP1, VP2 or VP3 are indicative of theAAV capsid heterogeneity).

In embodiments, the AAV particle includes an AAV1 capsid, an AAV2capsid, an AAV3 capsid, an AAV4 capsid, an AAV5 capsid, an AAV6 capsid,an AAV7 capsid, an AAV8 capsid, an AAVrh8 capsid, an AAV9 capsid, anAAV10 capsid, an AAV11 capsid, an AAV 12 capsid, or a variant thereof.

In embodiments, the masses of VP1, VP2, and VP3 are compared totheoretical masses of one or more of an AAV1 capsid, an AAV2 capsid, anAAV3 capsid, an AAV4 capsid, an AAV5 capsid, an AAV6 capsid, an AAV7capsid, an AAV8 capsid, an AAVrh8 capsid, an AAV9 capsid, an AAV10capsid, an AAV11 capsid, an AAV 12 capsid, or a variant thereof.

In embodiments, the AAV particle comprises an AAV1 ITR, an AAV2 ITR, anAAV3 ITR, an AAV4 ITR, an AAV5 ITR, an AAV6 ITR, an AAV7 ITR, an AAV8ITR, an AAVrh8 ITR, an AAV9 ITR, an AAV 10 ITR, an AAV11 ITR, or an AAV12 ITR.

In embodiments, the AAV particle has a capsid serotype selected fortransduction of cells of a subject's liver. In embodiments, the AAVparticle has a capsid serotype AAV7, AAV8, or AAV9, which are selectivefor the transduction of cells of a subject's liver.

In some embodiments, the AAV particle is a recombinant AAV (rAAV)particle. In some embodiments, the AAV particle comprises an AAV vectorencoding a heterologous transgene. In some embodiments, the AAV particlehas a capsid serotype AAV7, AAV8, or AAV9. In some embodiments, the AAVparticle has a capsid serotype AAV9. In some embodiments, the AAVparticle has a capsid serotype AAV9 and is a viral vector encodingLysosomal Alpha Glucosidase (GAA) linked to an antibody specific for anantigen expressed from a muscle cell (e.g., an anti-CD63 antibody).

While AAV was the model viral particle for this disclosure, it iscontemplated that the disclosed methods can be applied to profile avariety of viruses, e.g., the viral families, subfamilies, and genera.The methods of the present disclosure may find use, e.g., in profilingVPs to monitor VP expressions, posttranslational modifications, andtruncations and to ensure product consistency during VLP production, toconfirm site-direct mutagenesis or structural characterization forcapsid protein engineering applications, and/or to monitor or detectheterogeneity of a viral particle or preparation.

In embodiments, the viral vector encodes a heterologous transgene.

In embodiments, the viral particle belongs to a viral family selectedfrom the group consisting of Adenoviridae, Parvoviridae, Retroviridae,Baculoviridae, and Herpesviridae.

In embodiments, the viral particle belongs to a viral genus selectedfrom the group consisting of Atadenovirus, Aviadenovirus,Ichtadenovirus, Mastadenovirus, Siadenovirus, Ambidensovirus,Brevidensovirus, Hepandensovirus, Iteradensovirus, Penstyldensovirus,Amdoparvovirus, Aveparvovirus, Bocaparvovirus, Copiparvovirus,Dependoparvovirus, Erythroparvovirus, Protoparvovirus, Tetraparvovirus,Alpharetrovirus, Betaretrovirus, Deltaretrovirus, Epsilonretrovirus,Gammaretrovirus, Lentivirus, Spumavirus, Alphabaculovirus,Betabaculovirus, Deltabaculovirus, Gammabaculovirus, Iltovirus,Mardivirus, Simplexvirus, Varicellovirus, Cytomegalovirus,Muromegalovirus, Proboscivirus, Roseolovirus, Lymphocryptovirus,Macavirus, Percavirus, and Rhadinovirus.

In embodiments, the Retroviridae is Moloney murine sarcoma virus(MoMSV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumorvirus (MuMTV), gibbon ape leukemia virus (GaLV), feline leukemia virus(FLV), spumavirus, Friend virus, Murine Stem Cell Virus (MSCV) RousSarcoma Virus (RSV), human T cell leukemia viruses, HumanImmunodeficiency Viruse (HIV), feline immunodeficiency virus (FIV),equine immunodeficiency virus (EIV), visna-maedi virus; caprinearthritis-encephalitis virus; equine infectious anemia virus; felineimmunodeficiency virus (FIV); bovine immune deficiency virus (BIV); orsimian immunodeficiency virus (SIV).

Hydrophilic interaction chromatography (HILIC) is a variant of NP-HPLCthat can be performed with partially aqueous mobile phases, permittingnormal-phase separation of peptides, carbohydrates, nucleic acids, andmany proteins. The elution order for HILIC is least polar to most polar,the opposite of that in reversed-phase HPLC.

HILIC separates analytes based on polar interactions between theanalytes and the stationary phase (e.g., substrate). The polar analyteassociates with and is retained by the polar stationary phase.Adsorption strengths increase with increases in analyte polarity, andthe interaction between the polar analyte and the polar stationary phase(relative to the mobile phase) increases the elution time. Use of morepolar solvents in the mobile phase will decrease the retention time ofthe analytes, while more hydrophobic solvents tend to increase retentiontimes.

Various types of substrates can be used with HILIC, e.g., for columnchromatography, including silica, amino, amide, cellulose, cyclodextrinand polystyrene substrates. Examples of useful substrates, e.g., thatcan be used in column chromatography, include: polySulfoethylAspartamide (e.g., from PolyLC), a sulfobetaine substrate, e.g.,ZIC®-HILIC (e.g., from SeQuant), POROS® HS (e.g., from AppliedBiosystems), POROS® S (e.g., from Applied Biosystems), PolyHydroethylAspartamide (e.g., from PolyLC), Zorbax 300 SCX (e.g., from Agilent),PolyGLYCOPLEX® (e.g., from PolyLC), Amide-80 (e.g., from Tosohaas), TSKGEL® Amide-80 (e.g., from Tosohaas), Polyhydroxyethyl A (e.g., fromPolyLC), Glyco-Sep-N (e.g., from Oxford GlycoSciences), and AtlantisHILIC (e.g., from Waters). Columns that can be used in the disclosedmethods include columns that utilize one or more of the followingfunctional groups: carbamoyl groups, sulfopropyl groups, sulfoethylgroups (e.g., poly (2-sulfoethyl aspartamide)), hydroxyethyl groups(e.g., poly (2-hydroxyethyl aspartamide)) and aromatic sulfonic acidgroups.

In certain embodiments, the capsid proteins are separated on the HILICcolumn and subsequently eluted from the HILIC column, for example usinga mobile phase gradient to resolve the individual capsid proteins,thereby purifying and or separating capsid proteins in the sample. Incertain examples, the eluted capsid proteins from the HILIC column areseparated into one or more fractions. Such fractions can be used forsubsequent analysis, such as MS analysis. In certain embodiments, themethods include identifying capsid proteins present in one or more ofthe fractions.

The mobile phase used may include buffers with and without ion pairingagents, e.g., acetonitrile and water. Ion pairing agents includeformate, acetate, TFA and salts. Gradients of the buffers can be used,e.g., if two buffers are used, the concentration or percentage of thefirst buffer can decrease while the concentration or percentage of thesecond buffer increases over the course of the chromatography run. Forexample, the percentage of the first buffer can decrease from about100%, about 99%, about 95%, about 90%, about 85%, about 80%, about 75%,about 70%, about 65%, about 60%, about 50%, about 45%, or about 40% toabout 0%, about 1%, about 5%, about 10%, about 15%, about 20%, about25%, about 30%, about 35%, or about 40% over the course of thechromatography run. As another example, the percentage of the secondbuffer can increase from about 0%, about 1%, about 5%, about 10%, about15%, about 20%, about 25%, about 30%, about 35%, or about 40% to about100%, about 99%, about 95%, about 90%, about 85%, about 80%, about 75%,about 70%, about 65%, about 60%, about 50%, about 45%, or about 40% overthe course of the same run. In embodiments, the proportion of mobilephase A in the chromatography increases over time. Optionally, theconcentration or percentage of the first and second buffer can return totheir starting values at the end of the chromatography run. As anexample, the percentage of the first buffer can change in five stepsfrom 85% to 63% to 59% to 10% to 85%; while the percentage of the secondbuffer in the same steps changes from 15% to 37% to 41% to 90% to 15%.The percentages can change gradually as a linear gradient or in anon-linear (e.g., stepwise) fashion. For example, the gradient can bemultiphasic, e.g., biphasic, triphasic, etc. In preferred embodiments,the methods described herein use a decreasing acetonitrile buffergradient which corresponds to increasing polarity of the mobile phasewithout the use of ion pairing agents. In embodiments, the HILIC uses amobile phase A comprising trifluoroacetic acid in water. In embodiments,the mobile phase A comprises about 0.1% trifluoroacetic acid. Inembodiments, the chromatography comprises a mobile phase B comprisingtrifluoroacetic acid in acetonitrile. In embodiments, the mobile phase Bcomprises about 0.1% trifluoroacetic acid.

The column temperature can be maintained at a constant temperaturethroughout the chromatography run, e.g., using a commercial columnheater. In some embodiments, the column is maintained at a temperaturebetween about 50° C. to about 70° C., e.g., about 50° C. to about 60°C., about 55° C. to about 60° C., e.g., at about 50° C., about 55° C.,about 60° C., about 65° C., or about 70° C. In one embodiment, thetemperature is about 60° C.

The flow rate of the mobile phase can be between about 0 to about 100ml/min. For analytical proposes, flow rates typically range from 0 to 10ml/min, for preparative HPLC, flow rates in excess of 100 ml/min can beused. For example, the flow rate can be about 0.5, about 1, about 1.5,about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, or about 5ml/min. Substituting a column having the same packing, the same length,but a smaller diameter requires a reduction in the flow rate in order tomaintain the same retention time and resolution for peaks as seen with acolumn of wider diameter. Preferably, a flow rate equivalent to about 1ml/min in a 4.6×100 mm, 5 μm column is used.

The run time can be between about 15 to about 240 minutes, e.g., about20 to about 70 min, about 30 to about 60 min, about 40 to about 90 min,about 50 min to about 100 min, about 60 to about 120 min, about 50 toabout 80 min. In embodiments, the mobile phase A increases from about15% to about 100%, over about 45 minutes.

In some embodiments, the methods include subjecting a viral particle toliquid chromatography/mass spectrometry (LC/MS). As is known in the art,LC/MS utilizes liquid chromatography for physical separation of ions andmass spectrometry for generation of mass spectral data from the ions.Such mass spectral data may be used to determine, e.g., molecular weightor structure, identification of particles by mass, quantity, purity, andso forth. These data may represent properties of the detected ions suchas signal strength (e.g., abundance) over time (e.g., retention time),or relative abundance over mass-to-charge ratio.

In some embodiments, mass spectrometry (e.g., used in LC/MS as describedherein) may refer to electrospray ionization mass spectrometry (ESI-MS).ESI-MS is known in the art as a technique that uses electrical energy toanalyze ions derived from a solution using mass spectrometry (see, e.g.,Yamashita, M. and Fenn, J. B. (1984). Phys. Chem. 88:4451-4459). Ionicspecies (or neutral species that are ionized in solution or in gaseousphase) are transferred from a solution to a gaseous phase by dispersalin an aerosol of charged droplets, followed by solvent evaporation thatreduces the size of the charged droplets and sample ion ejection fromthe charge droplets as the solution is passed through a small capillarywith a voltage relative to ground (e.g., the wall of the surroundingchamberESI is performed by mixing the sample with volatile acid andorganic solvent and infusing it through a conductive needle charged withhigh voltage. The charged droplets that are sprayed (or ejected) fromthe needle end are directed into the mass spectrometer, and are dried upby heat and vacuum as they fly in. After the drops dry, the remainingcharged molecules are directed by electromagnetic lenses into the massdetector and mass analyzed. In one embodiment, the eluted sample isdeposited directly from the capillary into an electrospray nozzle, e.g.,the capillary functions as the sample loader. In another embodiment, thecapillary itself functions as both the extraction device and theelectrospray nozzle.

For MALDI, the analyte molecules (e.g., proteins) are deposited on metaltargets and co-crystallized with an organic matrix. The samples aredried and inserted into the mass spectrometer, and typically analyzedvia time-of-flight (TOF) detection. In one embodiment, the eluted sampleis deposited directly from the capillary onto the metal target, e.g.,the capillary itself functions as the sample loader. In one embodiment,the extracted analyte is deposited on a MALDI target, a MALDI ionizationmatrix is added, and the sample is ionized and analyzed, e.g., by TOFdetection.

In some embodiments, other ionization modes are used e.g. ESI-MS,turbospray ionization mass spectrometry, nanospray ionization massspectrometry, thermospray ionization mass spectrometry, sonic sprayionization mass spectrometry, SELDI-MS and MALDI-MS. In general, anadvantage of these methods is that they allow for the “just-in-time”purification of sample and direct introduction into the ionizingenvironment. It is important to note that the various ionization anddetection modes introduce their own constraints on the nature of thedesorption solution used, and it is important that the desorptionsolution be compatible with both. For example, the sample matrix in manyapplications must have low ionic strength, or reside within a particularpH range, etc. In ESI, salt in the sample can prevent detection bylowering the ionization or by clogging the nozzle. This problem isaddressed by presenting the analyte in low salt and/or by the use of avolatile salt. In the case of MALDI, the analyte should be in a solventcompatible with spotting on the target and with the ionization matrixemployed.

In some embodiments, the methods include subjecting a viral particle ofthe present disclosure, or subjecting digested fragments of a denaturedviral particle of the present disclosure, to liquid chromatography/massspectrometry-mass spectrometry (LC/MS/MS). As is known in the art,LC/MS/MS (the term “liquid chromatography-tandem mass spectrometry” maybe used interchangeably herein) utilizes liquid chromatography forphysical separation of ions and mass spectrometry for generation of massspectral data from the ions, where the mass spectrometry uses multiplestages of mass (e.g., m/z) separation, typically separated by afragmentation step. For example, ions of interest within a range of m/zmay be separated out in a first round of MS, fragmented, and thenfurther separated based on individual m/z in a second round of MS. Ionfragmentation may include without limitation a technique such ascollision-induced dissociation (CID), higher energy collisiondissociation (HCD), electron-capture dissociation (ECD), orelectron-transfer dissociation (ETD).

A variety of mass analyzers suitable for LC/MS and/or LC/MS/MS are knownin the art, including without limitation time-of-flight (TOF) analyzers,quadrupole mass filters, quadrupole TOF (QTOF), and ion traps (e.g., aFourier transform-based mass spectrometer or an Orbitrap). In Orbitrap,a barrel-like outer electrode at ground potential and a spindle-likecentral electrode are used to trap ions in trajectories rotatingelliptically around the central electrode with oscillations along thecentral axis, confined by the balance of centrifugal and electrostaticforces. The use of such instruments employs a Fourier transformoperation to convert a time domain signal (e.g., frequency) fromdetection of image current into a high resolution mass measurement(e.g., nano LC/MS/MS). Further descriptions and details may be found,e.g., in Scheltema, R. A. et al. (2014) Mol. Cell Proteomics13:3698-3708; Perry, R. H. et al. (2008) Mass. Spectrom. Rev.27:661-699; and Scigelova, M. et al. (2011) Mo/. Cell Proteomics 10:M111.009431.

In some embodiments, masses of viral capsid proteins may be determined,e.g., based on LC/MS and/or LC/MS/MS data. In some embodiments, massesof VP1, VP2 and VP3 of an AAV particle, or of fragments of VP1, VP2 andVP3 of the AAV particle, may be determined, e.g., based on LC/MS and/orLC/MS/MS data. Various methods to determine protein mass and/or identityfrom MS data are known in the art. For example, peptide massfingerprinting may be used to determine protein sequence based on MSdata, or proteins may be identified based on MS/MS data related to oneor more constituent peptides. When using tandem MS, product ion scanningmay be used to analyze m/z data related to one or more peptides of aprotein of interest. Software known in the art may then be used, e.g.,to match identified peaks to reference or known peaks, to group peaksinto isotopomer envelopes, and so forth. Peptide mass values may becompared to a database of known peptide sequences. For example, Mascotmay be used to match observed peptides with theoretical databasepeptides, e.g., resulting from application of a particular digestpattern to an in silico protein database. Other suitable software mayinclude without limitation Proteome Discoverer, ProteinProspector, X!Tandem, Pepfinder, Bonics, or MassLynx™ (Waters).

In some embodiments, the heterologous nucleic acid is operably linked toa promoter. Exemplary promoters include, but are not limited to, thecytomegalovirus (CMV) immediate early promoter, the RSV LTR, the MoMLVLTR, the phosphoglycerate kinase-1 (PGK) promoter, a simian virus 40(SV40) promoter and a CK6 promoter, a transthyretin promoter (TTR), a TKpromoter, a tetracycline responsive promoter (TRE), an HBV promoter, anhAAT promoter, a LSP promoter, chimeric liver-specific promoters (LSPs),the E2F promoter, the telomerase (hTERT) promoter; the cytomegalovirusenhancer/chicken beta-actin/Rabbit β-globin promoter (CAG promoter; Niwaet al., Gene, 1991, 108(2): 193-9) and the elongation factor 1- alphapromoter (EFI-alpha) promoter (Kim et al., Gene, 1990, 91(2):217-23 andGuo et al., Gene Ther., 1996, 3(9): 802-10). In some embodiments, thepromoter comprises a human β-glucuronidase promoter or a cytomegalovirusenhancer linked to a chicken β-actin (CBA) promoter. The promoter can bea constitutive, inducible or repressible promoter. In some embodiments,the invention provides a recombinant vector comprising a nucleic acidencoding a heterologous transgene of the present disclosure operablylinked to a CBA promoter.

Examples of constitutive promoters include, without limitation, theretroviral Rous sarcoma virus (RSV) LTR promoter (optionally with theRSV enhancer), the cytomegalovirus (CMV) promoter (optionally with theCMV enhancer), the SV40 promoter, the dihydrofolate reductase promoter,the 13-actin promoter, the phosphoglycerol kinase (PGK) promoter, andthe EFIa promoter.

Inducible promoters allow regulation of gene expression and can beregulated by exogenously supplied compounds, environmental factors suchas temperature, or the presence of a specific physiological state, e.g.,acute phase, a particular differentiation state of the cell, or inreplicating cells only. Inducible promoters and inducible systems areavailable from a variety of commercial sources, including, withoutlimitation, Invitrogen, Clontech and Ariad. Many other systems have beendescribed and can be readily selected by one of skill in the art.Examples of inducible promoters regulated by exogenously suppliedpromoters include the zinc-inducible sheep metallothionine (MT)promoter, the dexamethasone (Dex)-inducible mouse mammary tumor virus(MMTV) promoter, the T7 polymerase promoter system (WO 98/10088); theecdysone insect promoter (No et al., Proc. Natl. Acad. Sci. USA,93:3346-3351 (1996)), the tetracycline-repressible system (Gossen etal., Proc. Natl. Acad. Sci. USA, 89:5547-5551 (1992)), thetetracycline-inducible system (Gossen et al., Science, 268: 1766-1769(1995), see also Harvey et al., Curr. Opin. Chem. Biol., 2:512-518(1998)), the RU486-inducible system (Wang et al., Nat. Biotech.,15:239-243 (1997) and Wang et al, Gene Ther., 4:432-441 (1997)) and therapamycin-inducible system (Magari et al., J. Clin. Invest.,100:2865-2872 (1997)). Still other types of inducible promoters whichmay be useful in this context are those which are regulated by aspecific physiological state, e.g., temperature, acute phase, aparticular differentiation state of the cell, or in replicating cellsonly.

In another embodiment, the native promoter, or fragment thereof, for thetransgene will be used. The native promoter can be used when it isdesired that expression of the transgene should mimic the nativeexpression. The native promoter may be used when expression of thetransgene must be regulated temporally or developmentally, or in atissue-specific manner, or in response to specific transcriptionalstimuli. In a further embodiment, other native expression controlelements, such as enhancer elements, polyadenylation sites or Kozakconsensus sequences may also be used to mimic the native expression

In some embodiments, the regulatory sequences impart tissue-specificgene expression capabilities. In some cases, the tissue-specificregulatory sequences bind tissue-specific transcription factors thatinduce transcription in a tissue specific manner. Such tissue-specificregulatory sequences (e.g., promoters, enhancers, etc.) are well knownin the art.

In some embodiments, the vector comprises an intron. For example, insome embodiments, the intron is a chimeric intron derived from chickenbeta-actin and rabbit beta-globin. In some embodiments, the intron is aminute virus of mice (MVM) intron.

In some embodiments, the vector comprises a polyadenylation (polyA)sequence. Numerous examples of polyadenylation sequences are known inthe art, such as a bovine growth hormone (BGH) Poly(A) sequence (see,e.g., accession number EF592533), an SV40 polyadenylation sequence, andan HSV TK pA polyadenylation sequence.

The example systems, methods, and acts described in the embodimentspresented previously are illustrative, and, in alternative embodiments,certain acts can be performed in a different order, in parallel with oneanother, omitted entirely, and/or combined between different exampleembodiments, and/or certain additional acts can be performed, withoutdeparting from the scope and spirit of various embodiments. Accordingly,such alternative embodiments are included in the examples describedherein.

Although specific embodiments have been described above in detail, thedescription is merely for purposes of illustration. It should beappreciated, therefore, that many aspects described above are notintended as required or essential elements unless explicitly statedotherwise. Modifications of, and equivalent components or actscorresponding to, the disclosed aspects of the example embodiments, inaddition to those described above, can be made by a person of ordinaryskill in the art, having the benefit of the present disclosure, withoutdeparting from the spirit and scope of embodiments defined in thefollowing claims, the scope of which is to be accorded the broadestinterpretation so as to encompass such modifications and equivalentstructures.

The following examples are provided to illustrate particular features ofcertain embodiments. However, the particular features described belowshould not be considered as limitations on the scope of the invention,but rather as examples from which equivalents will be recognized bythose of ordinary skill in the art.

EXAMPLE

The following example is put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the methods of the invention, and is not intended to limitthe scope of what the inventors regard as their invention. Efforts havebeen made to ensure accuracy with respect to numbers used (e.g.,amounts, temperature, etc.) but some experimental errors and deviationsshould be accounted for. Unless indicated otherwise, parts are parts byweight, molecular weight is average molecular weight unless indicated,temperature is in degrees Centigrade, room temperature is about 25° C.,and pressure is at or near atmospheric.

Separation of Capsid Proteins From Intact AAV Particles

For separation of an intact AAV9 viral particles, 1 μL of the AAV samplewas injected onto a HILIC column, as described in the table below. Theresults of the separation are shown in FIG. 4. As shown, the three AAVcapsid proteins showed complete resolution from each other.

Table 1 shows a summary of the chromatographic conditions used for theseparation of AAV9 capsid proteins from an intact AAV9 capsid.

TABLE 1 Summary of chromatographic conditions UPLC System Waters ACQUITYUPLC I-Class Mobile Phase A: 0.1% TFA in water B: 0.1% TFA inacetonitrile Column ACQUITY UPLC ® Glycoprotein BEH Amide 1.7 μm, 2.1 mm× 150 mm, Part No. 186007963 Column 60° C. ± 1° C. TemperatureAutosampler  5° C. ± 2° C. Temperature Gradient Time Flow (min) (mL/min)% A % B 0 0.2 15.0 85.0 0.5 0.2 15.0 85.0 1.0 0.2 25.0 75.0 41.0 0.240.0 60.0 42.0 0.2 100.0 0.0 44.0 0.2 100.0 0.0 45.0 0.2 15.0 85.0 55.00.2 15.0 85.0 Detector Wavelength 280 nm

Compared to RP-based separation, the unique retention mechanism of theHILIC column worked surprisingly better for VP separation (see FIGS. 4,3A and 3B).

Mass Spectral Analysis of Separated AAV Capsid Proteins

One of the advantages of the methods described herein is that no samplepreparation is required. 1 uL of the sample was directly injected intothe into the LC-MS and the resulting data analyzed. The following tuneparameters were applied on a Q-Exactive Plus mass spectrometer forintact mass analysis:

Spray voltage 3.5 kV Capillary Temperature 350° C. S-lens RF level 60Sheath Gas flow rate 40 Aux Gas flow rate 15 In-source CID 0.0 eV m/zrange 800-4000

FIGS. 5A-5D show mass spectra of the individual capsid proteins. Asshown in FIG. 5A, there was protein heterogeneity in VP3.

As shown in FIG. 6, the peak height can be used to determine thestoichiometry of the intact AAV particles.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and theaccompanying figures. Such modifications are intended to fall within thescope of the appended claims.

What is claimed is:
 1. A method of determining the stoichiometry ofprotein components of an intact, non-enveloped viral particle,comprising: subjecting a sample comprising said viral particle tohydrophilic interaction liquid chromatography (HILIC) to separate theprotein components of the viral capsid of the viral particle;determining the masses of protein components of the viral capsid toidentify the protein components separated by HILIC; determining therelative abundance of the protein components of the viral capsid fromthe HILIC separation, thereby determining the stoichiometry of proteincomponents of the viral capsid of said viral particle.
 2. The method ofclaim 1, wherein the viral particle comprises an adeno-associated virus(AAV) particle.
 3. The method of claim 2, wherein the protein componentsof the protein capsid comprise VP1, VP2 and VP3 of the AAV particle. 4.The method of claim 2, wherein the masses of VP1, VP2, and VP3 arecompared to theoretical masses of one or more of an AAV1 capsid, an AAV2capsid, an AAV3 capsid, an AAV4 capsid, an AAV5 capsid, an AAV6 capsid,an AAV7 capsid, an AAV8 capsid, AAVrh8 capsid, an AAV9 capsid, an AAV10capsid, an AAV11 capsid, an AAV 12 capsid, or a variant thereof.
 5. Themethod of claim 3, wherein the AAV particle comprises an AAV1 capsid, anAAV2 capsid, an AAV3 capsid, an AAV4 capsid, an AAV5 capsid, an AAV6capsid, an AAV7 capsid, an AAV8 capsid, AAVrh8 capsid, an AAV9 capsid,an AAV10 capsid, an AAV11 capsid, an AAV 12 capsid, or a variantthereof.
 6. The method of claim 2, wherein the AAV particle comprises anAAV1 ITR, an AAV2 ITR, an AAV3 ITR, an AAV4 ITR, an AAV5 ITR, an AAV6ITR, an AAV7 ITR, an AAV8 ITR, an AAV9 ITR, an AAV 10 ITR, an AAV11 ITR,or an AAV 12 ITR.
 7. The method of claim 2, wherein the AAV particle isa recombinant AAV (rAAV) particle or an AAV vector encoding aheterologous transgene.
 8. The method of claim 2, wherein the AAVparticle has a capsid serotype selected for transduction of cells of asubject's liver.
 9. The method of claim 8, wherein the AAV particle hasa capsid serotype AAV7, AAV8, or AAV9.
 10. The method of claim 9,wherein the AAV particle has a capsid serotype AAV9.
 11. The method ofclaim 2, wherein the AAV particle has a capsid serotype AAV9 and is aviral vector encoding Lysosomal Alpha Glucosidase (GAA) linked to ananti-CD63 antibody.
 12. The method of claim 1, wherein the viralparticle comprises a viral vector encoding a heterologous transgene orbelongs to a viral family selected from the group consisting ofAdenoviridae, Parvoviridae, Retroviridae, Baculoviridae, andHerpesviridae.
 13. The method of claim 12, wherein the viral particlebelongs to a viral genus selected from the group consisting ofAtadenovirus, Aviadenovirus, Ichtadenovirus, Mastadenovirus,Siadenovirus, Ambidensovirus, Brevidensovirus, Hepandensovirus,Iteradensovirus, Penstyldensovirus, Amdoparvovirus, Aveparvovirus,Bocaparvovirus, Copiparvovirus, Dependoparvovirus, Erythroparvovirus,Protoparvovirus, Tetraparvovirus, Alpharetrovirus, Betaretrovirus,Deltaretrovirus, Epsilonretrovirus, Gammaretrovirus, Lentivirus,Spumavirus, Alphabaculovirus, Betabaculovirus, Deltabaculovirus,Gammabaculovirus, Iltovirus, Mardivirus, Simplexvirus, Varicellovirus,Cytomegalovirus, Muromegalovirus, Proboscivirus, Roseolovirus,Lymphocryptovirus, Macavirus, Percavirus, and Rhadinovirus.
 14. Themethod of claim 12, wherein the Retroviridae is Moloney murine sarcomavirus (MoMSV), Harvey murine sarcoma virus (HaMuSV), murine mammarytumor virus (MuMTV), gibbon ape leukemia virus (GaLV), feline leukemiavirus (FLV), spumavirus, Friend virus, Murine Stem Cell Virus (MSCV)Rous Sarcoma Virus (RSV), human T cell leukemia viruses, HumanImmunodeficiency Viruse (HIV), feline immunodeficiency virus (FIV),equine immunodeficiency virus (EIV), visna-maedi virus; caprinearthritis-encephalitis virus; equine infectious anemia virus; felineimmunodeficiency virus (FIV); bovine immune deficiency virus (BIV); orsimian immunodeficiency virus (SIV).
 15. The method of claim 1, whereinthe HILIC uses a mobile phase A comprising trifluoroacetic acid in wateror a mobile phase B comprising trifluoroacetic acid in acetonitrile. 16.The method of claim 13, wherein the mobile phase A or the mobile B phasecomprises about 0.1% trifluoroacetic acid.
 17. The method of claim 15,wherein the proportion of mobile phase A in the chromatography increasesover time.
 18. The method of claim 17, wherein mobile phase A increasesfrom about 15% to about 100%, over about 45 minutes.
 19. A method ofdetermining the heterogeneity of protein components of a capsid of anintact, non-enveloped viral particle comprising: subjecting a samplecomprising said viral particle to hydrophilic interaction liquidchromatography (HILIC) to separate protein components of the viralparticle's capsid; determining the masses of the protein components ofthe capsid; comparing the determined masses of the protein components ofthe viral particle's capsid with theoretical masses, wherein a deviationof one or more of the masses of protein components of the viralparticle's capsid from the theoretical masses is indicative of capsidheterogeneity.
 20. The method of claim 19, wherein the heterogeneitycomprises one or more of mixed serotypes, variant capsids, capsid aminoacid substitutions, truncated capsids, or modified capsids.